ABSTRACT Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector for therapeutic and vaccine applications, but its low transduction efficacy currently limits its utility. The primary reasons for low transduction efficacy are as follows: (i) inefficient trafficking of the rAAV genome to the nucleus, (ii) inefficient host cell-mediated synthesis of double-stranded DNA from the single-stranded genome, and (iii) Toll like receptor (TLR) 9 responses. The goal of this proposal is to identify small molecules that increase the transduction efficacy of rAAV transduction by modulating any one of the barriers. One immediate application will be the use of these compounds in the rAAV-based gene-therapy against HIV developed in Dr. Michael Farzan?s laboratory at The Scripps Research Institute. The rAAV vector expresses an antibody-like immunoadhesin (eCD4-Ig), a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, which binds to the HIV-1/simian immunodeficiency virus (SIV) envelope glycoprotein and protects macaques from infection. Increased transduction efficacy will decrease both the cost of treatment and the adaptive immune response to the viral capsid and expressed transgene that limits the use of rAAV vectors in humans. However, the significance of our studies goes beyond HIV-1 prophylaxis. These small molecules will be useful for other rAAV-based vaccines and therapeutics, such as vaccines developed for influenza, malaria, and dengue infection, or for the long-term delivery of biologics. In Phase I, we will (i) develop a high-throughput screening (HTS) assay to identify compounds that enhance rAAV transduction, (ii) identify transduction enhancers, (iii) broadly evaluate the mode of action, and prioritize hits for a Phase II study. Promising scaffolds will be subjected to a rational drug design program in Phase II, and will be advanced to IND enabling studies.